Transplantation of hematopoietic stem/progenitor cells (HSPC) is a required procedure for patients who have undergone high-dose chemotherapy and irradiation, and its efficiency depends on the homing ability of the intravenously administered HSPC. Homing of HSPC is a complex process strictly regulated by a multitude of factors.
HSPC trafficking represents one of the most important frontiers in clinical and experimental hematology. Transplantation of HSPC is widely used for the reconstitution of bone marrow hematopoiesis ablated by chemoradiotherapy. Trafficking of HSPC into the bone marrow is a complex and strictly regulated process which is controlled by a number of adhesion molecules, as well as by soluble factors, e.g., chemokines and the extracellular matrix (ECM).
Mature blood cells have a limited life span and have to therefore be constantly replenished by the committed, actively proliferating progenitors. While chemoradiotherapy eliminates dividing cells, including the pool of cycling HSPC, the recovery of mature blood cells following treatment requires a prolonged period of time and is generally accompanied by pancytopenia and bone marrow hypoplasia. Transplantation of HSPC is utilized to recover bone marrow hematopoietic activity after chemoradiotherapy, and its efficiency ultimately depends on the facility of HSPC homing. Homing of HSPC into the bone marrow is regulated by a vast variety of soluble and membrane-bound factors, including adhesion molecules, as well as cytokines, chemokines and interleukins, and ECM. The CD44/HA pathway is but one of the numerous cell signaling pathways mediating HSPC homing.
The generally used method of evaluation of HSPC homing is based on the lethal irradiation of animals followed by an intravenous administration of HSPC. After different periods of time (from 3 hours to 14 days, depending on the experimental design), the bone marrow and peripheral blood cells are harvested and examined for the number of HSPC using various assays (in vitro and in vivo clonogenic assays, FACS). It has been assumed that harvesting of the bone marrow early (3 hours) after the injection allows for the enumeration of “homed” cells (Vermulen M, et al (1998) Blood 92(3):894-900; Siminovich L, et al (1963) J Cell Comp Physiol 62:327; Hendrix P J, et al (1996) Exp Hemat 24:129-140; Oostendorp R A, et al (2000) Bone Marrow Transpl 26(5):559-556). However, because of the anatomical structure of the bone marrow, this technique does not make feasible the distinction of cells that have transmigrated into the extravascular marrow space from cells that have been arrested on the bone marrow vascular endothelium or are still rolling on its surface.
Another method for studying HSPC homing is based on the marrow repopulating ability (MRA) of HSPC injected into lethally irradiated recipients (Lord B I, et al. (1989) Exp Hematol 17:836; Visser J W M, Eliason J F. (1983) Cell Tissue Kinet. 16:385). In this model, bone marrow cells are harvested 2 weeks after bone marrow transplantation and the number of HSPCs is measured in CFU and CFUs assays. The assumption in this assay is that the amount of HSPC progeny directly correlates with the number of homed HSPC. Although this method gives a general understanding as to the involvement of selectively targeted cell surface molecules in the regulation of HSPC homing, it does not discriminate between the different phases of the homing cascade. Furthermore, because of the different turnover of cell surface receptors and the various antigen/antibody dissociation kinetics, effects of antibodies may be masked.
A recently proposed technique for studying HSPC homing is based on intravital microscopy of murine scalp bone marrow circulation (Mazo I, et al. (1998) J Exp Med 188(3):465-474). This technique provides for visualization of endothelial cell interactions under physiological flow conditions. Unfortunately, the technique does not permit long-term observation of these mice. Furthermore, using rodents to study migration of human HSPC may not authentically reflect the actual events in human bone marrow because many of the soluble and cell membrane associated molecules involved in regulating HSPC homing are species specific.
Approaches for studying cell migration also include static assays and assays under flow conditions. The phenotype and functions characteristics of endothelial cells in static conditions differ significantly from those under physiological flow. Static assays (adhesion and transwell assays) do not fairly represent HSPC-endothelial cell interactions occurring in vivo. Physiologic flow assays typically employ flow chambers. The existing flow chambers comprise a single compartment and provide a useful tool for examining tethering and adhesion of cells. However, there is no adequate device or method for studying the chemokine-mediated transmigration of cells under the conditions of flow. Currently, the only technique enumerating the number of migrated cells under the conditions of flow is based on the ability of the adherent cells to crawl beneath the cells that were grown on the glass slide (Cinamon G, et al (2001) J Leukoc Biol 69:860-866). This assay is monitored under the microscope using a high magnification objective, but is limited in that: (a) only a limited number of cells can be analyzed; (b) analysis is non-quantitative; (c) chemokine-mediated migration cannot be studied; (d) the microenvironment effects on endothelial cell function cannot be studied; and (e) there is no means for distinguishing between transmigrated cells and cells arrested in the endothelium.
Finally, none of the existing techniques permit manipulation of the local bone marrow microenvironment, and this limitation of these prior art techniques is crippling research into the role of the microenvironment in the regulation of endothelial cells function, which is becoming increasingly appreciated (Bautz F, et al (2000) Exp Hematol 28(6):700-706.). Although existing techniques have provided a large amount of important information, new methods are now required that will allow for visualizing each step of the homing cascade and for delineating mechanisms mediating HSPC homing: including endothelial cell, HSPC-ECM and HSPC-stromal cell interactions.